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Endothelial cells line the entire circulatory system, from the heart to the smallest capillary. In this protocol, attaining and maintaining vitality of the organ is critical. Minimizing the processing time between removing the organ and beginning the digestions will improve yields as cells are more viable and are not under hypoxic conditions for long. For this protocol to be most effective, mice should be 5-6 weeks of age. Older mice result in lower yields of endothelial cells. In our hands, as few as 5 mice can result in good yields.

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[Bio101] Isolation of Endothelial Cells from Mice

Cell Biology > Cell isolation and culture > Cell isolation
Author: Huan Pang
Huan PangAffiliation: Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, USA
For correspondence: pang_huan@hotmail.com
Bio-protocol author page: a48
4/20/2012, 10137 views, 4 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.205

[Abstract] Endothelial cells line the entire circulatory system, from the heart to the smallest capillary. In this protocol, attaining and maintaining vitality of the organ is critical. Minimizing the processing time between removing the organ and beginning the digestions will improve yields as cells are more viable and are not under hypoxic conditions for long. For this protocol to be most effective, mice should be 5-6 weeks of age. Older mice result in lower yields of endothelial cells. In our hands, as few as 5 mice can result in good yields.

Materials and Reagents

  1. Endothelial cells (EC)
  2. Endothelial cell growth supplement (ECGS) (Sigma-Aldrich, catalog number: E2759)
  3. Collagenase I (Life Technologies, InvitrogenTM, catalog number: 17100-017)
  4. Anti-CD31 antibody (BD Biosciences, PharmingenTM, catalog number: 550274)
  5. BSA
  6. DMEM
  7. Fetal calf serum (FCS)
  8. Fetal bovine serum (FBS)
  9. EDTA
  10. Phosphate buffered saline (PBS)
  11. Antibiotic
  12. Heparin
  13. F12 medium
  14. Isoflourane
  15. 70% ethanol
  16. Dynabeads
  17. M199
  18. Gelatin
  19. Pre-warmed digestion solution
  20. Digestion solution (see Recipes)
  21. Culture media (see Recipes)

Equipment

  1. Centrifuges
  2. Sterile culture hood
  3. Magnet

Procedure

  1. Harvesting of mouse lungs:
    1. Anesthetize the mice until drowzy. We usually use an inhaled anesthetic such as isoflurane.
    2. Sterilize the skin of the mice with 70% ethanol (sterilize instruments as well).
    3. Cut across the mouse, below but parallel to the diaphragm, and then cut up through the ribs to expose the thoracic cavity.
    4. Excise the lungs rapidly and place them in chilled DMEM on ice. Once all of the lungs have been collected, move to a sterile culture hood for the remainder of the procedure.
    5. Mince lung tissues, rinse several times to remove blood and incubate with 3 ml digestion solution at 37 °C for 45 min (3 x 15 min with pipetting between). Spin down.
    6. Mince the lung tissue finely (into pieces approximately 1 mm 3) and place into 5 ml of digestion buffer at 37 °C.
    7. Agitate the mixture constantly by hand, changing the digestion mixture every 5 min. To do this, spin down the fragments and remove the supernatant, replace the media using 5 ml of pre-warmed digestion solution and continue mixing.
    8. Add 1/10 volume of serum to the digestion solution. This will inactivate the enzymes and allow the cells to attach.
    9. Plate the cells on tissue culture plastic for 1 h. This pre-plate allows for the adhesion of fibroblasts but not endothelial cells (EC).
    10.  Remove the unattached cells and spin down at 200 x g for 5 min, then directly go to the enrichment protocol.

  2. Enriching for endothelial cells:
    Coat dynabeads with antibody to isolate EC: Add 25 μl of dynabeads (1 x 107) into a 1.5 ml centrifuge tube and wash the beads twice with 1 ml of PBS, add 100 μg anti-CD31 antibody in 100 μl PBS to dynabeads and incubate for 24 h at 4 °C with slow mixing. Collect the anti-CD31-coated dynabeads with the dynal magnet. Discard the supernatant while the tube is in the dynal magnet and wash the dynabeads with PBS once for 10 min at 4 °C, for 30 min at 4 °C and finally overnight at 4 °C. Collect the dynabeads using the magnet and discard the supernatant. Resuspend in 500 μl PBS/0.1% BSA and store at 4 °C at a final concentration of 2 x 107 beads/ml.

  3. Isolating endothelial cells using magnetic beads:
    1. After centrifuging the cells (200 x g for 5 min), add 15% FBS+M199. Wash the cells 3x with 15% FBS+M199 (37 °C), and centrifuge at 200 x g for 5 min to pellet cells.
    2. Resuspend in 1-2% FBS+M199 to a final concentration of 10-40 x 106 cells/ml.
    3. Add 1: 10 ratio of beads to endothelial cells (endothelial cells comprise approximately 0.02% of total cells).
    4. Rotate bead-pellet at 4 °C for 30 min. Place tube on magnet for 2-3 min, remove supernatant, and resuspend beads in 2 ml of M199. Repeat for a total of four washes.
    5. To liberate cells from anti-CD31 antibody coated beads, add 5 mM EDTA in PBS and rotate at 37 °C for 10 min.
    6. After 4 washes, place the beads in culture media. For culture of mouse EC, coat the plate with 0.2% gelatin for 20 min prior to plating cells.

Recipes

  1. Digestion solution
    1 mg/mI (250 U) collagenase I
    10 mg/mI BSA
    1 U/mI d ispase
    All dissolved in DMEM.
  2. Culture medium
    20% (v/v) FCS
    10 μg/ml ECGS
    100 U/ml antibiotic
    20 U/ml heparin
    All dissolved in F12 medium

References

  1. Dong, Q. G., Bernasconi, S., Lostaglio, S., De Calmanovici, R. W., Martin-Padura, I., Breviario, F., Garlanda, C., Ramponi, S., Mantovani, A. and Vecchi, A. (1997). A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants. Arterioscler Thromb Vasc Biol 17(8): 1599-1604.
  2. Sobczak, M., Dargatz, J. and Chrzanowska-Wodnicka, M. (2010). Isolation and culture of pulmonary endothelial cells from neonatal mice. J Vis Exp (46).


How to cite: Pang, H. (2012). Isolation of Endothelial Cells from Mice. Bio-protocol Bio101: e205. DOI: 10.21769/BioProtoc.205; Full Text



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12/21/2015 10:38:20 PM  

jie zhang
zhejiang university

Hello
I was wondering why you change M199 into F12 in culture medium? While M199 is suitable for endothelial cells.
Thank you.
Jie

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10/19/2013 11:15:08 PM  

Hua Li
UniSA

Hi ,

what is the meduim recipe? THank you very much.
and how many passages can do based on this protocol?

which passage will be good for tube-formation assay.

Thank you so much.

Hua

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10/8/2012 3:37:04 PM  

能留下你的电话或邮箱吗?我在做从小鼠中分离内皮细胞这个实验,想请教您,谢谢!

10/8/2012 10:42:30 PM  

Bio-protocol Editorial Team
bio-protocol.org

Thanks for being interested in this protocol. Unfortunately, it is out of reach for us to provide general consulting service. Here, we only are able to answer specific questions regarding this protocol. So, if you have questions regarding this protocol, please be specific.

Thanks,
Bio-protocol team

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2/2/2012 12:17:46 AM  

Hello,

I was wondering if you could tell me approximately how many endothelial cells you would get from a typical mouse lung. I'm trying to estimate how many endothelial cells are typically found in an adult and embryonic mouse.
Thanks so much!

Janet

2/3/2012 1:20:17 PM  

Huan Pang (Author)
Molecular Pharmacology,Albert Einstein College of Medicine

Hi,

Normally you could get aorund 10000~50000 endothelial cells from 5 mice in 5-6 weeks old. This is we roughly got in the experiment, but a lot of cells are lost during the isolation and purification precedure.

8/1/2014 9:53:12 AM  

BM H
UBC

Hello,

I find the very last steps somewhat confusing:
"4) Rotate bead-pellet at 4°C for 30 minutes. Place tube on magnet for 2-3minutes, remove supernatant, and resuspend beads in 2ml of M199. Repeat for a total of four washes.
5) To liberate cells from anti-CD31 antibody coated beads, add 5mM EDTA in PBS and rotate at37°C for 10 minutes.
6) After 4 washes, place the beads in culture media. For culture of mouse EC, coat the plate with 0.2% gelatin for 20 minutes prior to plating cells."

After washing the beads four times in step (4), you are liberating the cells with 5mM EDTA, so I assume the cells are now in the supernatant, and no longer attached to the beads. So why do you wash the beads again four more times in step (6) and then plate the beads in the culture medium?

Thank you.

Reply

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